CN113785046A

CN113785046A - Fermentation process

Info

Publication number: CN113785046A
Application number: CN202080029811.8A
Authority: CN
Inventors: P·德霍泰; R·科克斯; Q·祖尼
Original assignee: GlaxoSmithKline Biologicals SA
Current assignee: GlaxoSmithKline Biologicals SA
Priority date: 2019-02-22
Filing date: 2020-02-20
Publication date: 2021-12-10
Also published as: WO2020169703A1; CA3129649A1; JP2022521235A; EP3699263A1; BR112021015973A2; EP3927808A1; US20220169973A1; MX2021010154A

Abstract

The present invention relates to a process for the fermentation and production of virulence factors, particularly PT, of bordetella for large-scale production. More particularly, the method comprises a medium conditioning step performed prior to inoculation.

Description

Fermentation process

Background

Bordetella (B)Bordetella) Is a causative agent of many bacterial diseases, for example, Bordetella pertussis (also known as Haemophilus pertussis) is pertussisPertussis is a respiratory disease that may be severe in infants and young children. The clinical course of the disease is characterized by the onset of rapid coughing and subsequent inspiratory efforts often associated with a characteristic "rooming" sound. In severe cases, hypoxia can cause brain damage; however, the most common complication is secondary pneumonia.

Pertussis is generally considered to be caused by Bordetella pertussis (Bordetella pertussis)B. pertussis) Bordetella parapertussis (Bordetella parapertussis) caused, but sometimes isolated, from patients with typical signs and symptoms of pertussisB. parapertussis). Bordetella parapertussis infections are less frequent than Bordetella pertussis, with 5-10% of pertussis associated with Bordetella parapertussis (Mertsola (1985) Eur J Clin Microbiol 4: 123; Lautrop (1971) Lancet 1(7711): 1195-. Bordetella parapertussis is associated with mild clinical symptoms, which in combination with its serological cross-reactivity with Bordetella pertussis, makes Bordetella parapertussis difficult to diagnose.

The first generation vaccine against bordetella pertussis was a whole cell vaccine consisting of a whole killed bacterium. These were introduced in many countries in the 50 s of the 20 th century and the 60 s of the 20 th century and successfully reduced the incidence of pertussis. A problem with whole cell bordetella pertussis vaccines is the high reactogenicity associated with them. Acellular vaccines containing purified bordetella pertussis proteins have low reactogenicity and have been adopted for vaccination programs in many countries. Acellular vaccines containing Pertussis Toxin (PT), Filamentous Haemagglutinin (FHA) and, quite often, Pertactin (PRN) are widely used and provide effective protection against the severity of pertussis.

Bordetella virulence factors for these vaccines are produced by fermentation and isolation of the resulting virulence factors, but from Bordetella species: (Bordetellaspecies) are fastidious organisms that are difficult to grow at high concentrations (Doern, Clin. Infect. Dis. 2000, 30:166-Virulence factors of the genus tertbia, in particular Pertussis Toxin (PT).

There remains a need in the art to improve the efficacy of bordetella fermentation and virulence factor production, particularly PT production, for large-scale production. The present inventors have surprisingly found that a medium conditioning step performed prior to inoculation significantly improves several measures of fermentation performance of bordetella at large scale, including increased PT yield, increased biomass and reduced fermentation time.

Summary of The Invention

In a first aspect of the present invention, there is provided a method of producing a conditioned growth medium, comprising:

a) providing a growth medium;

b) maintaining the growth medium at a temperature of about 28 ℃ to about 35 ℃ for about 20 to 35 hours; and

c) optionally stirring and/or aerating the growth medium to produce about 10h^-1To about 130 h^-1The volumetric mass transfer coefficient (kLa) of the oxygen,

thereby providing the conditioned growth medium.

More particularly, a first aspect of the invention provides a method of producing a sterile conditioned growth medium comprising:

a) providing a sterile growth medium;

b) maintaining the sterile growth medium at a temperature of about 28 ℃ to about 35 ℃ for about 20 to 35 hours; and

c) stirring and/or aerating the sterile growth medium to produce about 10 hours^-1To about 130 h^-1The volumetric mass transfer coefficient (kLa) of the oxygen,

thereby providing the sterile conditioned growth medium.

In a second aspect of the invention, there is provided a conditioned growth medium produced by a method comprising the steps of:

a) providing a growth medium;

c) optionally stirring and/or aerating the growth medium to produce about 10h^-1To about 130 h^-1Volumetric mass transfer coefficient (kLa) of oxygen.

More particularly, a second aspect of the invention provides a sterile conditioned growth medium produced by a method comprising the steps of:

a) providing a sterile growth medium;

c) stirring and/or aerating the sterile growth medium to produce about 10 hours^-1To about 130 h^-1Volumetric mass transfer coefficient (kLa) of oxygen.

In a third aspect of the invention, there is provided a method of culturing a bordetella species comprising:

a) inoculating the conditioned growth medium of the second aspect with at least one bordetella cell to produce a bordetella culture; and

b) the culture of bordetella is maintained under conditions that increase biomass and/or produce at least one bordetella protein.

In a fourth aspect of the present invention, there is provided a process for producing a bordetella protein, comprising:

a) inoculating the conditioned growth medium of the second aspect with at least one bordetella cell to produce a bordetella culture;

b) maintaining the bordetella culture under conditions that produce at least one bordetella protein; and

c) isolating the at least one bordetella protein from the culture.

In a fifth aspect of the invention, there is provided an isolated bordetella protein produced by a process comprising the steps of:

c) isolating the at least one bordetella protein from the culture.

In a sixth aspect of the invention, there is provided an immunogenic composition comprising an isolated bordetella protein produced by a process comprising the steps of:

c) isolating the at least one bordetella protein from the culture.

Brief Description of Drawings

FIG. 1 changes in biomass (solid line) and oxygen consumption (point) during fermentation of Bordetella in conditioned (black) and unconditioned (grey) media.

FIG. 2 is a graph of four measures of evaluating conditioning process parameters for fermentation performance of Bordetella (A) PT content; (B) FHA content; (C) biomass; and (D) surface plots (surface plots) of experimental design for the effect of fermentation time. The results predict that the optimal conditioning parameters for PT production and biomass production are a temperature of 31.2 ℃ and about 90h^-1kLa was adjusted for 34.6 hours.

FIG. 3 validation of the regulatory parameters at 1L bioreactor scale. Right panel: the fermentation of Bordetella in conditioned medium (

methods

2, 3 and 4; see example 3 for details) produced PT levels greater than 10% higher than the fermentation in unconditioned medium. Left panel: the biomass content is not significantly affected by the temperature or kLa at the 1L bioreactor scale.

FIG. 4 Effect of adjusting the duration on PT content and biomass at 1L bioreactor scale. The upper diagram: medium conditioning for 32 hours and 56 hours resulted in an increase in PT content during fermentation compared to unconditioned medium>10 percent. The following figures: biomass increased at 32 hours and 56 hours.

FIG. 5 verification of optimal regulatory parameters at 20L bioreactor scale. Fermentations carried out in conditioned medium at optimal process parameters (31 ℃; 32 h; kLa 90 h-1) resulted in at least a 10% increase in PT yield compared to either unconditioned medium (NC) or suboptimal regulatory parameters.

FIG. 6 mean growth curve with error bars (standard deviation) after fermentation in small scale vessels (< 1L) using conditioned medium in 800L fermentor or media preparation tank.

FIG. 7 mean growth curves with error bars (standard deviation) after fermentation in small scale vessels (< 1L) using (A) medium vs unconditioned medium conditioned for 32 hours in a 800L fermentor or (B) medium vs unconditioned medium conditioned for 32 hours in a medium preparation tank.

FIG. 8 mean growth curves with error bars (standard deviation) after fermentation in small scale vessels (< 1L) using medium conditioned for 20 or 32 hours in the medium preparation tank.

Detailed description of the invention

The present invention is based on the unexpected observation that the addition of a medium conditioning step prior to inoculation significantly improves several measures of fermentation performance of bordetella, including increased yield of bordetella protein, increased biomass and reduced fermentation time. The step or method is carried out in particular using sterile growth medium. More particularly, the step or method is aseptic. Even more particularly, the step or method is a sterile method using a sterile growth medium. The term "sterile process" as used herein refers to methods and conditions for preventing contamination by excluding microorganisms.

The term "conditioning" as used herein refers to a process of conditioning and making a medium sterile prior to inoculation with bacteria, in other words in the absence of bacteria. Conditioning is a process that typically includes aeration and/or agitation phases of the sterile growth medium to improve the performance of the subsequent fermentation step. In particular, the sterile growth medium remains sterile for the duration of the conditioning process. Thus, preferably, the method for producing the conditioned growth medium of the invention is a sterile method. Preferably, the method of the invention is a sterile method of producing a sterile conditioned growth medium.

Accordingly, one aspect of the invention is a method of producing a conditioned growth medium comprising: providing a growth medium; maintaining the growth medium at a temperature of about 28 ℃ to about 35 ℃ for about 20 to 35 hours; and optionally stirring and/or aerating the growth medium to produce about 10h^-1To about 130 h^-1Thereby providing a conditioned growth medium. More particularly, the present invention is a method, particularly a sterile method, for producing a conditioned growth medium comprising: providing a sterile growth medium; maintaining the sterile growth medium at a temperature of about 28 ℃ to about 35 ℃ for about 20 to 35 hours; and optionally stirring and/or aerating the sterile growth medium to produce about 10h^-1To about 130 h^-1Thereby providing a conditioned growth medium. The conditioned growth medium produced by this method from sterile growth medium is itself sterile, i.e., free of independently replicating living organisms.

The growth medium can be any medium capable of supporting the growth of bordetella cells. In certain embodiments, the growth medium is chemically defined Stainer Scholte (SS) medium or modified Stainer Scholte Medium (MSS). The composition of Stainer Scholte medium is described in Cohen and Wheeler, American Journal of Public Health (1946) 36: 371-. The medium is a modified Stainer Scholte medium if it contains essentially the same concentration of medium components as the SS medium, but contains a modification of the concentration of 1 to 5 medium components, lacks 1 to 3 medium components or contains 1 to 20 additional medium components.

In a particular embodiment, the modified Stainer and Scholte medium comprises dimethyl-beta-cyclodextrin (e.g., about 1 g/L) and acid hydrolyzed casein (e.g., about 10 g/L). In additional embodiments, the modified Stainer and Scholte media comprise L-cysteine (e.g., about 40 mg/L) in place of L-cystine; an increased sodium L-glutamate concentration (e.g., about 11.84 g/L); reduced glutathione concentrations (e.g., about 150 mg/L); and/or reduced ascorbic acid concentration (e.g., about 400 mg/L). Thus, in some embodiments, the growth medium is modified Stainer Scholte Medium (MSS). In some embodiments, the growth medium is a modified Stainer and Scholte medium comprising about 1g/L of dimethyl-beta-cyclodextrin and about 10g/L of acid hydrolyzed casein. In some embodiments, the growth medium is a modified Stainer and Scholte medium comprising about 40mg/L of L-cysteine instead of L-cystine, about 11.84g/L of sodium L-glutamate, about 150mg/L of glutathione, and/or about 400mg/L of ascorbic acid (e.g., about 400 mg/L).

Compounds that affect the production of virulence factors by Bordetella pertussis are generally through modulationbvg(Bordetella virulence genes) loci function and can therefore be termedbvgModulators (see e.g. EP 2809343B). Thus, in some embodiments, the growth medium may comprise at least one selected from the group consisting of niacin, magnesium salts, sulfates, phosphates, carbonates, sucrose, proline, sodium ions at a concentration greater than 100mM, antifoaming agents, glutathione, and sulfur-containing amino acidsbvgA modulator. In some embodiments of the present invention, the substrate is,bvgthe regulator is nicotinic acid. In some embodiments, the growth medium is a modified Stainer Scholte medium comprising niacin.

Adjusting the process parameters of temperature, duration and kLa

In one aspect of the invention, conditioning is achieved by maintaining the sterile growth medium at a specified temperature for a specified period of time prior to use in fermentation. In one embodiment, the sterile growth medium is maintained at a temperature of about 28 ℃ to about 35 ℃. In another embodiment, the sterile growth medium is maintained at a temperature of about 29 ℃ to about 33 ℃, or about 30 ℃ to about 32 ℃. In particular embodiments, the sterile growth medium is maintained at about 29, 30, 31, 32, or 33 ℃. In further embodiments, the sterile growth medium is maintained at about 30.0, 30.2, 30.4, 30.6, 30.8, 31.0, 31.2, 31.4, 31.6, 31.8, or 32.0 ℃.

In certain embodiments, the sterile growth medium is maintained at the desired temperature for about 20 to 35 hours. In another embodiment, the sterile growth medium is maintained at the desired temperature for about 25 to 35 hours or about 30 to 35 hours. In particular embodiments, the sterile growth medium is maintained at the desired temperature, for example at about 31 ℃ for about 29, 30, 31, 32, 33, 34, or 35 hours. In a preferred embodiment, the sterile growth medium is maintained at about 31 ℃ for about 32 hours.

In certain embodiments, growth medium conditioning is performed at a scale of at least 10L, at least 100L, at least 800, or at least 1000L of growth medium. In particular embodiments, the growth medium conditioning is performed at a scale of about 10-100L, about 100-500L, about 500-1000L, about 1000-1500L, about 1500-2000L, about 1000-2500L, or about 1500-2500L.

In some embodiments, the methods of the invention require that the sterile growth medium be maintained at a constant kLa throughout the conditioning process. kLa, the oxygen volumetric mass transfer coefficient, is a measure of the rate of oxygen entry into the medium. The higher the kLa, the higher the rate of oxygen introduction into the medium. Several factors, including culture medium volume and composition, agitation (e.g., stirring), aeration, pressure, and temperature will affect the kLa of a particular growth medium preparation.

Oxygen can be introduced into the sterile growth medium by agitation (e.g., stirring) and/or aeration (blowing compressed air through the medium). If there are different oxygen concentrations in the air introduced into the medium, the flow rate should be adjusted to take this into account. For example, if a 100% oxygen supply is introduced into the medium, the flow rate is correspondingly lower. If a gas containing less oxygen than air is introduced into the medium, a higher flow rate can be applied. If aeration is achieved by bubbling compressed air through the culture medium, the compressed air is in particular sterile filtered via a filter, more particularly a filter with sufficiently small pores to prevent microorganisms or spores from entering the container (e.g. bioreactor, fermenter or culture medium preparation tank) with the air, preferably a filter with a cut-off value of about 0.2 μm to about 0.45 μm.

kLa can be measured using methods known in the art, for example as described in example 1 of US 2008/0193475. The method involves setting the bioreactor to conditions where the media volume, temperature, pressure, agitation and aeration of its kLa are to be measured, venting by replacing air with nitrogen (bubbling out), inletting by resuming air aeration and measuring pO₂The rate at which it returns to its steady state level.

By plotting the logarithm (100-pO) against time₂%) to calculate kLa. The angular coefficient of the straight part of the graph corresponds to-kLa. Typically, only 20% to 80% pO are considered₂The data of (1).

The kLa of the media conditioning step or process is affected by many factors, including the agitation rate and aeration flow rate of the media. A constant kLa may be maintained while, for example, decreasing the agitation speed of the medium and increasing the aeration rate, or vice versa. In one embodiment, both the agitation rate and aeration rate of the growth medium are constant during medium conditioning. In one embodiment, the growth medium is continuously stirred throughout the duration of the conditioning. In another embodiment, the growth medium is continuously aerated throughout the duration of the conditioning. In another embodiment, both stirring and aeration are performed continuously throughout the duration of the conditioning.

For example, in the range of about 10-200 h^-1、10-150 h^-1、10-100 h^-1、10-80 h^-1、10-50 h^-1、10-40 h^-1、10-30 h^-1、20-150 h^-1、20-100 h^-1、20-50 h^-1、20-60 h^-1、20-80 h^-1、20-30 h^-1、20-40 h^-1、30-60 h^-1、60-80 h^-1、60-150 h^-1Or 60-200 h^-1Growth medium conditioning was performed at kLa. In a particular embodiment, at about 10h^-1To about 130 h^-1About 60h^-1To about 130 h^-1Or about 90h^-1Growth under kLa ofAnd (5) adjusting the culture medium. In a preferred embodiment, the kLa of the growth medium is maintained at about 90h^-1。

For a volume of 10-30 liters, for example, 10-30 h are achieved by using an air flow rate or aeration rate of 1-5 liters/min and an agitation rate of 200-400 rpm (revolutions per minute), for example an aeration rate of 2-4 liters/min and an agitation rate of 250-350 rpm^-1kLa of (1).

For a volume of 30-250 liters, for example, 30-60 h are achieved by using an air flow rate of 15-25 liters/min and an agitation speed of 150-^-1kLa of (1).

In a specific embodiment, at about 31 ℃, for about 32h, about 90h^-1Growth medium conditioning was performed at kLa.

The invention further provides a conditioned growth medium produced by the method of the invention. The conditioned growth medium is sterile. "sterile" is intended to mean that the growth medium is free or substantially free of bacterial cells, e.g., prior to inoculation with bordetella cells.

Accordingly, in one aspect, the present invention provides a conditioned growth medium produced by a method, particularly an aseptic method, comprising the steps of:

a) providing a sterile growth medium;

In particular, in step c), the sterile growth medium is continuously stirred and/or aerated for the duration of step b). In some embodiments, in step c), the sterile growth medium is continuously stirred for the duration of step b). In other embodiments, in step c), the sterile growth medium is continuously aerated for the duration of step b). In some embodiments, in step c), the sterile growth medium is continuously stirred and aerated for the duration of step b).

Fermentation of bordetella

In one aspect, the invention provides a method of culturing a bordetella species comprising: inoculating a conditioned growth medium produced as described herein with at least one bordetella cell to produce a bordetella culture; and maintaining the bordetella culture under conditions that increase biomass and/or produce at least one bordetella protein. In particular, the conditioned growth medium is sterile prior to inoculation with the at least one bordetella cell.

In another aspect of the present invention, there is provided a method for producing a bordetella protein, comprising:

a) inoculating a conditioned growth medium produced as described herein with at least one bordetella cell to produce a bordetella culture;

c) isolating the at least one bordetella protein from the culture.

Preferably, the process for producing a bordetella protein comprises:

a) inoculating a sterile conditioned growth medium produced as described herein with at least one bordetella cell to produce a bordetella culture;

c) isolating the at least one bordetella protein from the culture.

In a further aspect of the invention, there is provided an isolated bordetella protein produced by a process comprising the steps of:

c) isolating the at least one bordetella protein from the culture.

Preferably, the isolated bordetella protein is produced by a process comprising the steps of:

c) isolating the at least one bordetella protein from the culture.

In some embodiments, the bordetella species is bordetella pertussis or bordetella parapertussis. In one embodiment, the at least one Bordetella protein is selected from the group consisting of Pertussis Toxin (PT), Filamentous Hemagglutinin (FHA), pertactin (PRN; also known as 69K), and Adenylate Cyclase (AC). In a preferred embodiment, the at least one bordetella protein is a pertussis toxin, e.g., genetically detoxified pertussis toxin (PTg). In some embodiments, the pertussis toxin is a genetically detoxified pertussis toxin in which two catalytic residues of the S1 subunit (Arg 9 and Glu 129) are mutated to Lys9 and Gly129 (referred to as PT-9K/129G mutants).

Can be determined by measuring the Optical Density (OD) (also called OD) at 650 nm, for example₆₅₀) To quantify biomass. In one embodiment, the bacterial density measured at 650 nm at the end of the fermentation reaches at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60 or at least 70 OD units. Optical density may also be expressed in absorbance units (A.U.). End of fermentation is defined as dissolved oxygen (pO) in the culture₂) The time at which the minimum is reached and the ramp begins.

In another embodiment, the invention provides an immunogenic composition comprising an isolated bordetella protein produced by the method of the invention. The immunogenic compositions of the invention may further comprise one or more pharmaceutically acceptable excipients, adjuvants and/or additional antigens.

The conditioned growth medium prepared according to the process of the present invention provides certain advantages in the fermentation of bordetella cultures. For example, the methods of the invention can result in the production of at least one bordetella protein in a yield of at least 5%, 10%, 15%, 20%, 25%, or 30% greater than that produced by the same method performed on an unconditioned growth medium. In a specific embodiment, the yield of PT is increased by at least 10% compared to the yield produced by the same method performed on a non-conditioned growth medium, and the yield of filamentous hemagglutinin is unchanged or higher. The unconditioned growth medium used for comparison is a growth medium which has not been treated or conditioned according to the method of the invention, for example a freshly prepared growth medium. One skilled in the art will recognize that the conditioned and unconditioned growth media used for comparison are of the same type, i.e., are capable of homogeneous comparison.

In another embodiment, fermentation time-is defined as from inoculation to pO₂The time when the level reaches a minimum and begins to climb-is at least 10% shorter than the fermentation time of the same process performed with unconditioned growth medium.

In another embodiment, the bordetella fermentation process of the invention produces at least 10% more biomass at the end of fermentation than the same process conducted with unconditioned growth medium.

In another embodiment, the bordetella fermentation process of the invention further comprises the step of purifying one or more bordetella proteins from the bordetella culture.

In one embodiment, the present invention provides an aseptic process for producing a conditioned growth medium comprising: providing sterile modified Stainer and Scholte growth media; subjecting the sterile growth medium to a temperature of about 30-32 deg.CHolding for about 31-33 hours; and agitating and/or aerating the sterile growth medium to produce about 90 hours^-1Thereby providing a conditioned growth medium. More particularly, an aseptic process for producing a conditioned growth medium comprising: providing sterile modified Stainer and Scholte growth media; maintaining the sterile growth medium at a temperature of about 30-32 ℃ for about 31-33 hours; and agitating and aerating the sterile growth medium to produce about 90 hours^-1Thereby providing a conditioned growth medium.

In one embodiment, the present invention provides an aseptic process for producing a conditioned growth medium comprising: providing a sterile modified Stainer and Scholte growth medium comprising about 1g/L dimethyl-beta-cyclodextrin, about 10g/L acid hydrolyzed casein, about 40mg/L L-cysteine instead of L-cystine, about 11.84g/L L-sodium glutamate; about 150mg/L glutathione and about 400mg/L ascorbic acid; maintaining the sterile growth medium at a temperature of about 31 ℃ for about 32 hours; and agitating and/or aerating the sterile growth medium to produce about 90 hours^-1Thereby providing a conditioned growth medium. More particularly, an aseptic process for producing a conditioned growth medium comprising: providing a sterile modified Stainer and Scholte growth medium comprising about 1g/L dimethyl-beta-cyclodextrin, about 10g/L acid hydrolyzed casein, about 40mg/L L-cysteine instead of L-cystine, about 11.84g/L L-sodium glutamate; about 150mg/L glutathione and about 400mg/L ascorbic acid; maintaining the sterile growth medium at a temperature of about 31 ℃ for about 32 hours; and agitating and aerating the sterile growth medium to produce about 90 hours^-1Thereby providing a conditioned growth medium.

Description of the preferred embodiments

Embodiment 1. a method of producing a conditioned growth medium comprising: (i) providing a growth medium; (ii) maintaining the growth medium at a temperature of about 28 ℃ to about 35 ℃ for about 20 to 35 hours; and (iii) optionally stirringMixing and/or aerating the growth medium to produce about 10 hours^-1To about 130 h^-1Thereby providing a conditioned growth medium.

Embodiment 2. the process of embodiment 1, wherein step b) is carried out at a temperature of from about 29 ℃ to about 33 ℃, from about 30 ℃ to about 32 ℃, or about 31 ℃.

Embodiment 3. the method of embodiments 1-2, wherein step b) is performed for about 25 to 35 hours, about 30 to 35 hours, or about 32 hours.

Embodiment 4. the method of embodiments 1-3, wherein step c) comprises continuously stirring the growth medium for the duration of step b).

Embodiment 5 the method of embodiment 4, wherein the stirring is effected for about 60h^-1To about 130 h^-1Or about 90h^-1At a stirring speed of the oxygen volume mass transfer coefficient (kLa).

Embodiment 6. the method of embodiments 1-5, wherein step c) comprises continuously aerating the growth medium for the duration of step b).

Embodiment 7 the method of embodiment 6, wherein said aeration is effected for about 60 hours^-1To about 130 h^-1Or about 90h^-1At a flow rate of the oxygen volume mass transfer coefficient (kLa).

Embodiment 8 the method of embodiments 1-7, wherein step c) comprises continuously stirring and aerating the growth medium for the duration of step b).

Embodiment 9 the process of embodiment 8, wherein the stirring and aeration is carried out for about 60 hours^-1To about 130 h^-1Or about 90h^-1The oxygen volume mass transfer coefficient (kLa) of the reactor.

Embodiment 10. the method of embodiments 1-9, wherein the method is performed on a scale of at least 10L, at least 100L, or at least 1000L of growth medium.

Embodiment 11. a conditioned growth medium produced by the method of embodiments 1-10.

Embodiment 12 a method of culturing a bordetella species comprising: (i) inoculating the conditioned growth medium of embodiment 11 with at least one bordetella cell to produce a bordetella culture; and (ii) maintaining the bordetella culture under conditions that increase biomass and/or produce at least one bordetella protein.

Embodiment 13. a process for producing a bordetella protein comprising: (i) inoculating the conditioned growth medium of embodiment 11 with at least one bordetella cell to produce a bordetella culture; (ii) maintaining the bordetella culture under conditions that produce at least one bordetella protein; and (iii) isolating the at least one bordetella protein from the culture.

Embodiment 14. the method of embodiments 12-13, wherein the at least one bordetella protein is selected from the group consisting of pertussis toxin, filamentous hemagglutinin, pertactin and adenylate cyclase.

Embodiment 15. the process of embodiments 12-14, wherein the at least one bordetella protein is produced in a yield of at least 10% greater than the yield produced by the same process performed on unconditioned growth medium.

Embodiment 16 the method of embodiments 12-15 wherein the at least one bordetella protein is a pertussis toxin, e.g., a genetically detoxified pertussis toxin.

Embodiment 17 the method of embodiments 12-16 wherein the at least one bordetella protein is a genetically detoxified pertussis toxin wherein the two catalytic residues of the S1 subunit (Arg 9 and Glu 129) are mutated to Lys9 and Gly 129.

Embodiment 18 the method of embodiment 16, wherein the production of filamentous hemagglutinin is unchanged or higher compared to the production produced by the same method performed with a non-conditioned growth medium.

Embodiment 19. the process of embodiments 12-18, wherein the fermentation time is at least 10% shorter than the fermentation time of the same process carried out with non-conditioned growth medium.

Embodiment 20. the process of embodiments 12-19, wherein the biomass of the bordetella culture at the end of fermentation is at least 10% higher than that produced by the same process performed with unconditioned growth medium.

Embodiment 21. the process of embodiments 12-20 wherein the process further comprises the step of purifying one or more bordetella proteins from the bordetella culture.

Embodiment 22. the method of embodiments 1-10, wherein the growth medium is sterile.

Embodiment 23. an isolated bordetella protein produced by the method of embodiments 12-22.

Embodiment 24 an immunogenic composition comprising the isolated bordetella protein of embodiment 23.

Embodiment 25. an aseptic process for producing a conditioned growth medium comprising: (i) providing a sterile growth medium; (ii) maintaining the sterile growth medium at a temperature of about 28 ℃ to about 35 ℃ for about 20 to 35 hours; and (iii) agitating and/or aerating the sterile growth medium to produce about 10 hours^-1To about 130 h^-1Thereby providing a conditioned growth medium.

Embodiment 26 the sterile process of embodiment 25, wherein step b) is carried out at a temperature of about 29 ℃ to about 33 ℃, about 30 ℃ to about 32 ℃, or about 31 ℃.

Embodiment 27. the sterile process of embodiment 25 or 26, wherein step b) is carried out for about 25 to 35 hours, about 30 to 35 hours, or about 32 hours.

Embodiment 28 the sterile method of embodiment 25, 26 or 27, wherein step c) comprises continuously stirring said sterile growth medium for the duration of step b).

Embodiment 29 the sterile method of embodiment 28, wherein the stirring is effected for about 60h^-1To about 130 h^-1Or about 90h^-1At a stirring speed of the oxygen volume mass transfer coefficient (kLa).

Embodiment 30 the sterile method of embodiments 25 to 29, wherein step c) comprises continuously aerating the sterile growth medium for the duration of step b).

Embodiment 31 the sterile process of embodiment 30, wherein the aeration is effected for about 60 hours^-1To about 130 h^-1Or about 90h^-1At a flow rate of the oxygen volume mass transfer coefficient (kLa).

Embodiment 32 the sterile process of embodiments 25 to 31, wherein step c) comprises continuously stirring and aerating the sterile growth medium for the duration of step b).

Embodiment 33 the sterile process of embodiment 32, wherein the stirring and aeration is effected for about 60 hours^-1To about 130 h^-1Or about 90h^-1The oxygen volume mass transfer coefficient (kLa) of the reactor.

Embodiment 34 the sterile method of embodiments 25 to 33, wherein the method is performed on a scale of at least 10L, at least 100L, or at least 1000L of sterile growth medium.

Embodiment 35. a conditioned growth medium produced by the sterile process of embodiments 25 to 34.

Embodiment 36 a method of culturing a bordetella species comprising: (i) inoculating the conditioned growth medium of embodiment 35 with at least one bordetella cell to produce a bordetella culture; and (ii) maintaining the bordetella culture under conditions that increase biomass and/or produce at least one bordetella protein.

Embodiment 37. a process for producing a bordetella protein comprising: (i) inoculating the conditioned growth medium of embodiment 35 with at least one bordetella cell to produce a bordetella culture; (ii) maintaining the bordetella culture under conditions that produce at least one bordetella protein; and (iii) isolating the at least one bordetella protein from the culture.

Embodiment 38 the method of embodiment 36 or 37, wherein said at least one bordetella protein is selected from the group consisting of pertussis toxin, filamentous hemagglutinin, pertactin and adenylate cyclase.

Embodiment 39. the method of embodiments 36, 37 or 38 wherein the at least one bordetella protein is produced in a yield of at least 10% greater than the yield produced by the same method performed on unconditioned growth medium.

Embodiment 40 the method of embodiments 36 to 39, wherein said at least one bordetella protein is a pertussis toxin, e.g., a genetically detoxified pertussis toxin.

Embodiment 41 the method of embodiments 36 to 40, wherein said at least one bordetella protein is a genetically detoxified pertussis toxin wherein the two catalytic residues of the S1 subunit (Arg 9 and Glu 129) are mutated to Lys9 and Gly 129.

Embodiment 42 the method of embodiment 40 or 41, wherein the production of filamentous hemagglutinin is unchanged or higher compared to the production produced by the same method performed with a non-conditioned growth medium.

Embodiment 43 the process of embodiments 36 to 42, wherein the fermentation time is at least 10% shorter than the fermentation time of the same process carried out with a non-conditioned growth medium.

Embodiment 44. the process of embodiments 36 to 43, wherein the biomass of the bordetella culture at the end of fermentation is at least 10% higher than the biomass produced by the same process carried out with unconditioned growth medium.

Embodiment 45 the process of embodiments 36 to 44 wherein the process further comprises the step of purifying one or more bordetella proteins from the bordetella culture.

Embodiment 46. the method of any preceding embodiment, wherein the growth medium is modified Stainer Scholte Medium (MSS) optionally comprising niacin.

Embodiment 47 the method of embodiment 46, wherein the growth medium is a modified Stainer and Scholte medium comprising about 1g/L dimethyl-beta-cyclodextrin and about 10g/L acid hydrolyzed casein.

Embodiment 48. the method of embodiment 46 or 47, wherein the growth medium is a medium comprising about 40mg/L L-cysteine instead of L-cystine; about 11.84g/L L-sodium glutamate; about 150mg/L glutathione; and/or about 400mg/L ascorbic acid (e.g., about 400 mg/L) of modified Stainer and Scholte media.

Embodiment 49 an aseptic process for producing a sterile conditioned growth medium comprising: (a) providing a sterile growth medium; (b) maintaining the sterile growth medium at a temperature of about 29 ℃ to about 33 ℃, about 30 ℃ to about 32 ℃, or about 31 ℃ for about 25 to 35 hours, about 30 to 35 hours, or about 32 hours; and (c) continuously stirring and/or aerating the sterile growth medium for the duration of step b) to produce about 60 hours^-1To about 130 h^-1Or about 90h^-1(ii) oxygen volumetric mass transfer coefficient (kLa), thereby providing a sterile conditioned growth medium; wherein the growth medium is modified Stainer Scholte Medium (MSS) optionally comprising niacin.

General rule

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise.

In addition, the numerical limits given for the concentrations or amounts of materials, such as the concentrations of the components of the solution or the ratios thereof, and the reaction conditions, such as temperature, pressure, and cycle time, are intended to be approximate. The term "about" as used herein is intended to mean an amount of ± 10%. Unless the context requires otherwise, the word "between" when used in reference to a numerical range (e.g., "between X and Y" or "between about X and about Y") is intended to include the endpoints of that range (i.e., including X and Y).

The term "comprising" means "including". Thus, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated compound or composition (e.g. nucleic acid, polypeptide, antigen) or step or group of compounds or steps but not the exclusion of any other compound, composition, step or group thereof. The term "consisting of …" means "including and limited to". The term "consisting essentially of …" means that the composition or method may include additional ingredients and/or steps, but only if the additional ingredients and/or steps do not materially alter the basic and novel characteristics of the claimed composition or method. The abbreviation "e.g. (e.g.)" is derived from latin-exempli gratia and is used herein to denote non-limiting examples. The abbreviation "e.g." is therefore synonymous with the term "e.g. (for example)".

The invention will be further described with reference to the following non-limiting examples and the accompanying drawings.

Examples

Example 1 validation of the Regulation Effect of the Medium on a laboratory Scale

Preliminary observations indicate that yield changes in commercial scale bordetella fermentations may be attributed to differences in growth medium regulation prior to inoculation. To explore the source of this change, the effect of growth medium conditioning prior to inoculation was examined in a laboratory scale model approach.

Laboratory scale model

A laboratory scale model was developed to reproduce the three steps of the commercial process: 1) a pre-culture sequence (pre-culturetrain); 2) adjusting a culture medium; and 3) fermenting. The preculture sequence steps refer to preculture steps for accumulating sufficient biomass for inoculation of the fermentation step. Since medium conditioning is a sterile process step that is carried out in the absence of bacteria, medium conditioning is carried out independently of the pre-culture sequence steps, e.g., before, after, or in parallel. In preculture, use 10⁹Bordetella pertussis CFU inoculation contains 30ml of fresh medium (MSS; medium derived from Stainer and Scholte J. Gen. Microbinary. 63:211-1g/L of arginine and acid hydrolysis of casein 10g/L, replacement of L-cystine by 40mg/L of L-cysteine, and use of a first shake flask pre-culture of higher concentration of sodium L-glutamate (11.84 g/L), reduced glutathione (150 mg/L) and ascorbic acid (400 mg/L)) and incubation at 35 ℃ for 24 hours with agitation at 150 rpm. The first preculture was used to inoculate a second shake flask preculture containing 1000ml of fresh Medium (MSS). The second preculture was incubated at 35 ℃ for 24 hours with stirring at 150 rpm. An aliquot of the pre-culture sequence was then used to inoculate the conditioned growth medium for fermentation as described below.

Media conditioning was performed in parallel with the pre-culture sequence. 1 liter of sterile growth medium (of the same type used for the preculture) was aseptically transferred into a 1 liter bioreactor (BioBlock platform (4X 1L bioreactor), Eppendorf) and aerated at 35 ℃ with an air flow of 20L of air per hour, with a stirring speed of 430 rpm (60 h)^-1kLa) for 40 hours. As a control, the unconditioned growth medium was maintained at 4 ℃ without further processing. The preparation of conditioned and unconditioned media was carried out four times using the same procedure (Prep 1, Prep 2, Prep 3, Prep 4).

By inoculating conditioned and unconditioned media with a preculture sequence of an inoculum of Bordetella and fermenting in a small scale fermentation vessel: (<1L) under standard conditions (35 ℃, at least 90 h)^-1kLa) and the fermentation step is performed on a small scale.

Measurement of fermentation Performance

Evaluation of fermentation performance as indicated by biomass, PT production and fermentation time was repeated at least 3 times for each preparation. Record biomass, dissolved oxygen pressure (pO) throughout fermentation₂) And on-line measurement of pH. The start of fermentation is defined as the time at which the bordetella preculture sequence is added to the fermentation vessel. End of fermentation is defined as dissolved oxygen (pO)₂) Reach a minimum and begin to climb back to 100% of the time point. pO₂The inflection point of (a) indicates that the carbon source in the culture is depleted and the cells are transitioning from the growth phase to the stationary phase. The fermentation time is thus the start and the end of the fermentationThe amount of time between the end of the fermentation.

At the end of the fermentation, the supernatant was recovered by centrifugation (14000 g, 10min RT), filtered (0.22 um sieve) and stored at-20 ℃ for further analysis. Pertussis Toxin (PT) content at the end of fermentation was determined by ELISA using standard methods.

Results

The results are summarized in table 1 as the average percent change [ condition/non-condition-1 (%) ] relative to the non-conditioned medium. For all four replicates (Preps 1-4), pertussis toxin content at the end of fermentation increased by at least 10% in conditioned medium compared to unconditioned medium. For three of these preparations (Preps 2-3), the biomass at the end of the fermentation in the conditioned medium increased correspondingly compared to the non-conditioned medium. Finally, when the culture was grown in conditioned medium, a tendency was observed to decrease the fermentation time compared to non-conditioned medium.

TABLE 1

。

The growth kinetics of Prep 2 are shown in fig. 1 as representative examples of these four preparations. The bordetella cell growth (biomass) in conditioned medium (black solid line) and unconditioned medium (grey solid line) was similar during the first 15-20 hours of culture. Thereafter, the growth rate of the cells in the conditioned medium is higher than that in the unconditioned medium. As biomass increased, dissolved oxygen decreased, and a more rapid decrease was observed in the conditioned medium fermentations. At the end of the fermentation, indicated by a rapid re-increase in dissolved oxygen, the biomass is significantly higher in the conditioned medium fermentation conditions than in the unconditioned fermentation conditions.

Results of initial small scale studies indicate that prior medium regulation has a positive effect on bordetella cell growth and PT production. In particular, for fermentations carried out in conditioned media, a significant increase in the biomass and PT content at the end of the fermentation was observed. A trend of shorter fermentation times in conditioned media was also observed. The results of the laboratory scale model confirm that observations made at commercial scale indicate that the effect of the modulation is independent of the scale of the fermentation. No significant effect of medium adjustment on the pH of the medium was observed during cell growth (data not shown).

Without being bound by a fundamental theory, the observed medium-conditioning effects may be related to biochemical modifications that occur during medium conditioning, such as oxidation of one or more medium components, that improve cell growth and virulence factor productivity during the fermentation step.

Example 2 identification of Process parameters affecting the Regulation of the Medium

To better determine the regulatory parameters that lead to improved fermentation performance of bordetella, a design of experiments (DoE) study was conducted.

Method

The effect of three regulatory process parameters on subsequent fermentation performance was evaluated in a core composite design with 3 levels (min/core/max) of each parameter as shown in table 2. The process parameters are the regulation temperature, the regulation duration and the oxygen volumetric mass transfer coefficient (kLa), which are factors of the aeration flow rate and the stirring speed.

TABLE 2 media Conditioning Process parameters

Parameter(s)	Minimum size	Center of a ship	Maximum of
				Temperature (. degree.C.)	28	35	40
kLa (h^-1)	10	50	90
				Duration (hours)	3	23	43

DoE was performed in 60 runs (runs) performed over 6 weeks (see table 3). Weekly, 3 x 1L conditioning bioreactors were charged with 1L of sterile growth medium freshly prepared as described in example 1 and conditioned at 3 different kLa "temperatures. During the conditioning process, each conditioning bioreactor was sampled at 3 different time points (3 h, 23h and 43 h) to examine the effect of the conditioning duration. Samples of the medium extracted at 3 and 23h were immediately stored at 4 ℃ to stabilize the samples. After the last sample was taken, 9 media samples from 3 bioreactors were transferred to small scale fermentation vessels (< 1L) and inoculated with the bordetella preculture sequence prepared as described in example 1 to evaluate growth and antigen production.

Fermentation performance indicators were evaluated by measuring PT and FHA content (ELISA), biomass content (growth curve and final optical density) and fermentation time (measured as described in example 1).

Results

The results are shown in table 3 and fig. 2. The influence of the variation of the adjusting process parameters on the fermentation performance indexes is different. The longer the duration of the medium conditioning, the better the biomass and PT content obtained. FHA levels are not affected by increased duration of conditioning. Adjusting the temperature affects PT production but not growth performance (biomass). Finally, changes in kLa affect biomass but not PT production.

The results of DoE predicted the design space for the values of the regulatory parameters associated with increased PT (at least 10%) relative to the unconditioned medium (fig. 2A-D). The model also predicts that the optimal tuning parameters for PT production and biomass production are temperature at 31.2 ℃ and about 90h^-1kLa was adjusted for 34.6 h.

TABLE 3 results of the experimental design

NC-unconditional

NT not tested。

Example 3 validation of the parameters of the Regulation at the 1 liter bioreactor Scale

To validate the medium conditioning design space identified in example 2 for 10% increased PT yield, medium conditioning and fermentation processes were performed at a 1L bioreactor scale using conditioning parameters within the predicted design space.

Method

The platform of the 4X 1L-bioreactor (BioBlock, Eppendorf) was used for medium conditioning before inoculation. This platform allows parallel adjustment of 4 media preparations and continuous evaluation of their fermentation performance. For the conditioning step, 1L of sterile growth medium (see examples 1 and 2) was aseptically transferred to each bioreactor and subjected to the process parameters described in the experimental design (below). If the experimental design requires unconditioned medium, one of the four bioreactors remains empty during the conditioning step to allow for later transfer of the unconditioned medium (see below).

When a predetermined duration of medium conditioning is reached, the conditioning is stopped. For some procedures, 1 liter (1L) of unconditioned medium (prepared as in examples 1 and 2) was aseptically transferred to one of four bioreactors. The following conditions were used to calibrate the 100% Dissolved Oxygen (DO) level in each bioreactor: temperature (35 ℃), atmospheric pressure, air flow rate (2L of air blown per minute) and stirring speed (300 rpm or revolutions per minute). 300 μ L of antifoam (Simethicone 15%) was added aseptically to each bioreactor.

Inoculation was achieved by adding 150 mL of bordetella pertussis inoculum (prepared in parallel with the conditioning procedure as described in examples 1 and 2). During the fermentation, the temperature (35 ℃) was kept at a constant level. Foam control during fermentation was performed by adding an antifoam (Simethicone 1.5%). The dissolved oxygen level was set at 35% to compensate for the head pressure applied during larger scale fermentations and adjusted by increasing agitation when the DO dropped below 35%. The minimum stirring speed was set to 300 rpm; the maximum stirring speed was set to 1100 rpm. The pH was adjusted to 7.2 by the addition of acetic acid 50% (w/v or weight/volume).

At the end of the fermentation (as defined in example 1), the biomass yield was determined by measuring the optical density and by adjusting the total amount of acetic acid added by pH (the latter being evaluated as an orthogonal method for biomass content). Pertussis Toxin (PT) production in culture supernatants was determined by ELISA using standard methods.

Design of experiments

In example 2, the design space that results in at least a 10% increase in PT yield was calculated. In this experiment, we compared the fermentation performance (fermentation time, biomass, PT and FHA yield) of media conditioned at 3 sets of different process parameters (methods 2-4 in table 4) within the calculated PT design space relative to unconditioned media (method 1 in table 4).

Methods

2 and 3, respectively, tested at 90h^-1Or 60h^-1The optimum operating parameters for PT production (32 h at 31 ℃ C.). Method 4 test of the tempering temperature at 35 ℃ and 60h^-1Operating parameters at kLa. This experiment was conductedIs carried out once.

TABLE 4

Method	Duration (hours)	Temperature (. degree.C.)	kLa (h^-1)
				1	32	4	0
2	32	31	90
				3	32	31	60
4	32	35	60

Results

As shown in figure 3, fermentations carried out in conditioned media (

methods

2, 3 and 4) produced PT levels greater than 10% higher than fermentations carried out in unconditioned media (right panel). The biomass content was not significantly affected by temperature or kLa at the 1L bioreactor scale (left panel). These results validate the design space for 10% increased PT yield identified in the experimental design study (example 2).

Example 4 Effect of duration of Conditioning on a 1 liter bioreactor Scale

To explore the effect of the conditioning duration over a wider time frame, a conditioning temperature of 31 ℃ was used for 90h^-1K La value of and<adjustment duration of 3h, 32h or 56h example 3 (table 5) was repeated. The experiment was performed in duplicate.

TABLE 5

Method	Duration (hours)	Temperature (. degree.C.)	kLa (h^-1)
				5	32	4	0
6	<3	31	90
				7	32	31	90
8	56	31	90

Results

As shown in FIG. 4, medium adjustments for 32h and 56h resulted in an increase in PT content compared to unconditioned medium>10% (upper panel). Biomass also increased at 32h and 56h (lower panel). The effect of increased duration of conditioning on PT content and biomass reached a plateau around 32 h.

Example 5 verification of optimal regulatory parameters at 20 liter bioreactor Scale

Optimal media conditioning parameters were also verified in bordetella fermentations conducted at 20L bioreactor scale.

Method

20L fermentors (Biolafite ™) were used for medium conditioning prior to inoculation. 10L of sterile growth medium prepared as in example 1 was aseptically transferred to a 20L bioreactor and subjected to the adjusted process parameters outlined in Table 6.

TABLE 6

。

Preculture sequences were prepared as described in example 1, except that the first and second precultures were prepared in duplicate (2X 30mL of the first preculture; 2X 1000mL of the second preculture). After 24 h (+/-1h) growth at 35 ℃ (+/-1 ℃) and 150 rpm, the two disposable flasks from the second preculture were pooled. Once the second preculture had stopped, the pooled preculture was used to inoculate the fermenter.

Upon reaching the predetermined duration of the conditioning step, conditioning was stopped and the following conditions were used to calibrate 100% Dissolved Oxygen (DO) levels prior to inoculation: temperature (35 ℃), head pressure (0.4 bar), air flow rate (14.6L of air blown per minute) and stirring speed (50 rpm or revolutions per minute). 3 mL of antifoam (Simethicone 15%) was added aseptically to each bioreactor.

Inoculation was achieved by adding 1.5L of pooled preculture. During the fermentation, the temperature (35 ℃) and the head pressure (0.4 bar) were kept at constant levels. Foam control during fermentation was performed by adding an antifoam (Simethicone 1.5%). The dissolved oxygen level was set at 25% and adjusted by increasing agitation when the DO dropped below 25%. The minimum stirring speed was set to 50 rpm; the maximum stirring speed was set to 1000 rpm. The pH was adjusted to 7.2 by the addition of acetic acid 50% (w/v or weight/volume).

Design of experiments

The experiment was designed to compare the optimum process parameters (31 ℃, 32h, 90 h)^-1kLa) and the fermentation performance of a medium adjusted with only one parameter different from the optimal process parameters.

At week 1, the effect of the optimal parameters relative to the unconditioned medium (scheme 1) was verified (scheme 2). At

weeks

2 and 3, the effect of low kLa regulation (scheme 3-low aeration and scheme 5-low agitation speed) was compared with the optimal operating conditions (schemes 4 and 6). The effect of low temperature (run 7: 23 ℃) and low duration (run 9: 3 h) was compared to the optimal operating parameters (runs 8 and 10) at

weeks

4 and 5, respectively.

Results

As shown in figure 5 and table 7, fermentations performed in conditioned media with optimal process parameters yielded PT yields 10% higher than these:

-unconditioned medium (week 1)

Medium conditioned at low kLa (. apprxeq.10 h-1) due to Low aeration flow rate (week 2)

Medium conditioned at low kLa (. apprxeq.10 h-1) due to low stirring speed (week 3)

Medium conditioned at Low temperature (23 ℃) (week 4)

Medium conditioned with short duration (3 h) (week 5)

These data confirm the optimal operating parameters for media conditioning for PT production at 20L fermentation scale. No negative effects on biomass yield, FHA yield and fermentation time were observed. Surprisingly, although the small scale studies described in examples 1-4 did not find the effect of low kLa on PT yield, the negative effect of low kLa was observed at the 20L fermentation scale. Thus, duration, temperature and kLa all appear to be important factors for producing a regulatory effect at the 20L fermentation scale.

TABLE 7 fermentation Performance on 20L Scale

NC, non-conditioned growth medium.

Example 6 verification of optimal tuning parameters at Large Scale

Media conditioning parameters were performed at large scale and verified in bordetella fermentations performed in small scale fermentation vessels (< 1L).

Method

The 800L fermentor and 2400L media preparation tank were used for large-scale media conditioning prior to inoculation. Sterile growth medium as prepared in example 1 was aseptically transferred to the fermentor (800L) or media preparation tank (2400L) and subjected to the following media to adjust the process parameters (table 7). Due to differences between vessels, such as aerated sparger design and agitation systems, Kla values achieved in media preparation tanks were lower than those obtained in fermenters.

TABLE 7

Unconditioned growth medium (NC) -control.

Samples of 5 sterile conditioned media were collected in Novaseptum sampling bags at different time points and stored immediately at 4 ℃.

Each of the 5 samples was transferred to a small scale fermentation vessel (< 1L) and inoculated with a bordetella preculture sequence prepared as described in example 1 to evaluate growth and antigen production.

Results

As shown in FIG. 6, the effect of medium conditioning on growth performance during the subsequent fermentation step is comparable regardless of the type of vessel used to perform the conditioning step. However, in each case, the use of pre-conditioned medium had a demonstrable positive effect on the fermentation (FIGS. 7(A) and (B)).

The bacteria grew faster compared to the non-conditioned medium (control), meaning that the total fermentation time could be reduced by an average of about 8%. Using conditioned medium, the final biomass achieved was higher than the biomass of the control (approximately 9% higher at the beginning of the stationary phase). Interestingly, the growth performance of the conditioned media over 20 and 32 hours was comparable (fig. 8).

Fermentation in conditioned medium improved the yield of PT and FHA. Specifically, PT productivity was increased by 7% after medium conditioning for 20 hours and by 15% after medium conditioning for 32 hours relative to the control. Similarly, FHA productivity increased by 9% after medium conditioning for 20 hours and by 15% after medium conditioning for 32 hours compared to controls.

TABLE 8 summary results of growth performance and antigen productivity. The fermentation time and the biomass at the beginning of the stationary phase are determined directly from the growth curve.

a-the value at the beginning of the stationary phase, determined from the mean growth curve;

b-OD read on samples stored at 4 ℃ for 2 days after the end of fermentation.

These data demonstrate the positive effect of using a cell-free medium conditioning step at large scale (up to 2400L) before the conditioned medium is used for bacterial fermentation.

Claims

1. A method of producing a conditioned growth medium comprising:

a. providing a sterile growth medium;

b. maintaining the sterile growth medium at a temperature of about 28 ℃ to about 35 ℃ for about 20 to 35 hours; and

c. stirring and/or aerating the sterile growth medium to produce about 10 hours^-1To about 130 h^-1The volumetric mass transfer coefficient (kLa) of the oxygen,

thereby providing the conditioned growth medium.

2. The process of claim 1, wherein step b) is carried out at a temperature of from about 29 ℃ to about 33 ℃, from about 30 ℃ to about 32 ℃, or about 31 ℃.

3. The method of claims 1-2, wherein step b) is performed for about 25 to 35 hours, about 30 to 35 hours, or about 32 hours.

4. The method of claims 1-3, wherein in step c), the sterile growth medium is continuously stirred for the duration of step b).

5. The method of claim 4, wherein the agitation is for about 60 hours^-1To about 130 h^-1Or about 90h^-1At a stirring speed of the oxygen volume mass transfer coefficient (kLa).

6. The method of claims 1-3, wherein in step c), the sterile growth medium is aerated continuously for the duration of step b).

7. The method of claim 6, wherein said aeration is for about 60 hours^-1To about 130 h^-1Or about 90h^-1At a flow rate of the oxygen volume mass transfer coefficient (kLa).

8. The method of claims 1-3, wherein step c) comprises continuously stirring and aerating the sterile growth medium for the duration of step b).

9. The method of claim 8, wherein said stirring and aerating occurs for about 60 hours^-1To about 130 h^-1Or about 90h^-1The oxygen volume mass transfer coefficient (kLa) of the reactor.

10. The method of claims 1-9, wherein the method is performed on a scale of at least 10L, at least 100L, at least 800L, or at least 1000L of sterile growth medium.

11. The method according to any of the preceding claims, wherein the growth medium is modified Stainer Scholte Medium (MSS) optionally comprising niacin.

12. The method of claim 11, wherein the growth medium is a modified Stainer and Scholte medium comprising about 1g/L dimethyl-beta-cyclodextrin and about 10g/L acid hydrolyzed casein.

13. The method of claim 11 or 12, wherein the growth medium is a medium comprising about 40mg/L L-cysteine in place of L-cystine; about 11.84g/L L-sodium glutamate; about 150mg/L glutathione; and/or about 400mg/L ascorbic acid (e.g., about 400 mg/L) of modified Stainer and Scholte media.

14. The method of any one of claims 1 to 13, wherein the method is aseptic.

15. A sterile conditioned growth medium produced by the method of any one of claims 1-14.

16. A method of culturing a bordetella species comprising:

a. inoculating the sterile conditioned growth medium of claim 15 with at least one bordetella cell to produce a bordetella culture; and

b. the culture of bordetella is maintained under conditions that increase biomass and/or produce at least one bordetella protein.

17. A process for producing a bordetella protein comprising:

a. inoculating the sterile conditioned growth medium of claim 15 with at least one bordetella cell to produce a bordetella culture;

b. maintaining the bordetella culture under conditions that produce at least one bordetella protein; and

c. isolating the at least one bordetella protein from the culture.

18. The method of claim 16 or 17, wherein the at least one bordetella protein is selected from the group consisting of pertussis toxin, filamentous hemagglutinin, pertactin and adenylate cyclase.

19. The process of claim 16, 17 or 18, wherein the at least one bordetella protein is produced in a yield of at least 10% greater than the yield produced by the same process performed with non-conditioned growth medium.

20. The method of claims 16 to 19, wherein the at least one bordetella protein is a pertussis toxin, such as a genetically detoxified pertussis toxin.

21. The method of claim 20, wherein the at least one bordetella protein is a genetically detoxified pertussis toxin wherein two catalytic residues of the S1 subunit (Arg 9 and Glu 129) are mutated to Lys9 and Gly 129.

22. The method of claims 16 to 21, wherein the at least one bordetella protein is or further comprises a filamentous hemagglutinin.

23. The method of claim 22, wherein the production of filamentous hemagglutinin is unchanged or higher compared to the production produced by the same method performed with a non-conditioned growth medium.

24. The process of claims 16 to 23, wherein the fermentation time is at least 10% shorter than the fermentation time of the same process performed with an unconditioned growth medium.

25. The process of claims 16 to 24, wherein the biomass of the bordetella culture at the end of fermentation is at least 10% higher than that produced by the same process carried out with non-conditioned growth medium.

26. The process of claims 16 to 25, wherein the process further comprises the step of purifying one or more bordetella proteins from the bordetella culture.

27. An isolated bordetella protein produced by the process of claims 16 to 26.

28. An immunogenic composition comprising the isolated bordetella protein of claim 27.